Methods of selecting retinal pigmented epithelial cells

ABSTRACT

A method of selecting retinal pigmented epithelial (RPE) cells from a mixed population of cells is disclosed. The method comprises:
         (a) analyzing the cells of the mixed population of cells for at least one of the following parameters:   (i) cells which autofluorescence above a predetermined threshold;   (ii) cells which express CD81 above a predetermined threshold; and   (iii) cells which scatter light perpendicular to a laser beam above a predetermined threshold; and   (b) selecting cells which are positive for at least one of the parameters, thereby sorting RPE cells from a mixed population of cells.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof selecting retinal pigmented epithelial cells (RPE cells).

The retinal pigmented epithelium (RPE) is a monolayer of pigmentedcells, which lies between the neural retina and the choriocapillars. TheRPE cells play crucial roles in the maintenance and function of theretina and its photoreceptors. These include the formation of theblood-retinal barrier, absorption of stray light, supply of nutrients tothe neural retina, regeneration of visual pigment, and uptake andrecycling of shed outer segments of photoreceptors.

Retinal tissue may degenerate for a number of reasons. Among them are:artery or vein occlusion, diabetic retinopathy and retinopathy ofprematurity, which are usually hereditary. Diseases such as retinitispigmentosa, retinoschisis, lattic degeneration, Best disease, and agerelated macular degeneration (AMD) are characterized by progressivetypes of retinal degeneration.

RPE cells may potentially be used for cell replacement therapy of thedegenerating RPE in retinal diseases mentioned above. It may be alsoused as a vehicle for the introduction of genes for the treatment ofretinal degeneration diseases. These cells may also serve as an in vitromodel of retinal degeneration diseases, as a tool for high throughputscreening for a therapeutic effect of small molecules, and for thediscovery and testing of new drugs for retinal degeneration diseases.RPE cells could also be used for basic research of RPE development,maturation, characteristics, properties, metabolism, immunogenicity,function and interaction with other cell types.

Human fetal and adult RPE has been used as an alternative donor sourcefor allogeneic transplantation. However, practical problems in obtainingsufficient tissue supply and the ethical concerns regarding the use oftissues from aborted fetuses limit widespread use of these donorsources. Given these limitations in supply of adult and fetal RPEgrafts, the potential of alternative donor sources have been studied.Human pluripotent stem cells provide significant advantages as a sourceof RPE cells for transplantation. Their pluripotent developmentalpotential may enable their differentiation into authentic functional RPEcells, and given their potential for infinite self renewal, they mayserve as an unlimited donor source of RPE cells. Indeed, it has beendemonstrated that human embryonic stem cells (hESCs) and human inducedpluripotent stem cells (iPS) may differentiate into RPE cells in vitro,attenuate retinal degeneration and preserve visual function aftersubretinal transplantation to the Royal College of Surgeons (RCS) ratmodel of retinal degeneration that is caused by RPE dysfunction.Therefore, pluripotent stem cells may be an unlimited source for theproduction of RPE cells.

Current protocols for the derivation of RPE cells from pluripotent stemcells yields mixed populations of pigmented and non-pigmented cells.However, pure populations of pigmented cells are desired for the usageof RPE cells in basic research, drug discovery and cell therapy. So farclusters of RPE cells have been identified as areas of dark pigmentationwithin cultures of multiple subtypes of differentiated cells and weremechanically dissected and isolated on the basis of their pigmentation.This approach is operator dependent, work intensive, depends onsubjective judgment, inconsistent, inaccurate and can not provide purepopulations of pigmented cells.

Klimanskaya et al [Methods in Enzymology, Vol. 418, p.169-194, 2006]teaches that it is difficult to dissociate RPE cells followingdifferentiation and accordingly sorting by FACS is not recommended.

Additional background art relevant to the present invention includesRowland et al., J. Cell. Physiol. 227: 457-466, 2012 and Shi et al Stemcell Research and Therapeutics, Chapter I, pages 1-24.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of selecting retinal pigmented epithelial(RPE) cells from a mixed population of cells, comprising:

(a) analyzing the cells of the mixed population of cells for at leastone of the following parameters:

(i) cells which autofluorescence above a predetermined threshold;

(ii) cells which express CD81 above a predetermined threshold; and

(iii) cells which scatter light perpendicular to a laser beam above apredetermined threshold; and

(b) selecting cells which are positive for at least one of theparameters, thereby sorting RPE cells from a mixed population of cells.

According to some embodiments of the present invention the RPE cells arecharacterized by expression of at least one marker selected from thegroup consisting of bestrophin, CRALBP, MITF and RPE65.

According to some embodiments of the present invention the RPE cells aregenerated by ex-vivo differentiating pluripotent stem cells towards anRPE lineage.

According to some embodiments of the present invention the pluripotentstem cells comprise embryonic stem cells (ESCs).

According to some embodiments of the present invention the pluripotentstem cells comprise induced pluripotent stem (iPS) cells.

According to some embodiments of the present invention the ex vivodifferentiating is effected by culturing the pluripotent stem cells in amedium comprising at least one member of the transforming growth factorβ (TGFβ) superfamily.

According to some embodiments of the present invention the mediumfurther comprises nicotinamide (NA).

According to some embodiments of the present invention the culturingcomprises a first culture stage in a first medium comprisingnicotinamide (NA), the first medium being devoid of the at least onemember of the TGFβ superfamily and a second culture stage in a secondmedium comprising NA and the at least one member of the TGFβsuperfamily.

According to some embodiments of the present invention, the firstculture stage is effected for at least two days. According to someembodiments of the present invention the at least one member of the TGFβsuperfamily is selected from the group consisting of TGFβ1, TGFβ3 andactivin A.

According to some embodiments of the present invention the selectingcells is performed using a fluorescence activated cell sorter (FACS).

According to some embodiments of the present invention the selectingcells which autofluoresce is performed using a 780/60 nm filter of aFACS.

According to some embodiments of the present invention the selectingcells which express CD81 is effected using an antibody which binds toCD81.

According to some embodiments of the present invention the antibodycomprises a detectable moiety.

According to some embodiments of the present invention the methodfurther comprises dispersing the mixed population of cells prior to theselecting.

According to some embodiments of the present invention the dispersing isperformed using trypsin.

According to some embodiments of the present invention the methodfurther comprises culturing the RPE cells following the selecting in amedium comprising nicotinamide.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-B: Single-cell suspensions were produced frommechanically-separated pigmented and non-pigmented clusters and analyzedby FACS as described in Materials and Methods. Dot plot analysis ofunstained cells shows that the pigmented clusters are enriched with anautofluorescent cell-population, detected by the FL3 (780/60 nm) filter.This population was not detected in the non-pigmented clusters.

FIGS. 2A-B: Single-cell suspensions were produced frommechanically-separated pigmented and non-pigmented clusters and analyzedby FACS, as described in Materials and Methods. Dot plot FSC/SSCanalysis shows that the pigmented clusters are enriched with a high SSCvalue population, relative to the cells from the non-pigmented clusters.

FIG. 3: Single-cell suspensions were produced frommechanically-separated pigmented and non-pigmented clusters and stainedwith FITC-labeled anti-CD81 antibody. FACS analysis of these cells showsthat the pigmented clusters express high levels of CD81, relative to thenon-pigmented clusters, as reflected by their mean fluorescenceintensity (MFI).

FIGS. 4A-D: FACS sorting was carried out as described in Materials andMethods. Sorting gates were based on forward and side light scatter (FSCand SSC, respectively), as well as FL2 and FL3 parameters. This figurepresents FACS analysis before (“pre-sort mix” dot plot, FIG. 4A) andafter FACS sorting (“Post-sort SSC low”, FIG. 4B: “Post-sort SSC high”;FIG. 4C and “Post-sort autofluorescent”, FIG. 4D) dot plots. FSC/SSC dotplots are shown on the left column, demonstrating the location of “SSChigh” and “SSC low” gates. The “Autofluorescent” gate is based on FL2(576/26 nm) filter and the FL3 (780/60 nm) filter dot plots, which areshown on the right column.

FIGS. 5A-C: Light microscopy of post-sorting cells of the“Autofluorescent”, “SSC high” and “SSC low” populations, one dayfollowing the sorting and plating on 0.1% gelatine. Distinct cellmorphologies can be identified at different sorted populations:dark-pigmented cytoplasm in the “Autofluorescent” sorted population,light and non-granulated cytoplasm in the “SSC low” sorted populationand morphologies of both types in the “SSC high” sorted population.

FIGS. 6A-C: Light microscopy of post-sorting cells of the“Autofluorescent”, “SSC high” and “SSC low” populations eight daysfollowing the sorting and plating on gelatine. Distinct cellmorphologies can be identified at the different sorted populations. Thecells in the “Autofluorescent” sorted population acquire polygonal shapeand dark cytoplasm, with high cell density, which implies relativelyhigh proliferation rate. The “SSC low” sorted population have non-RPEmorphology. The “SSC high” sorted population yields cells of both celltypes.

FIGS. 7A-C: Light microscopy of post-sorting cells of the“Autofluorescent”, “SSC high” and “SSC low” populations, 16 daysfollowing the sorting and plating on gelatine. Significant differenceswere found in cell morphology of the “SSC low” population, in comparisonwith the “autofluorescent” and “SSC high” progeny. The cells in the“Autofluorescent” sorted population had polygonal shapes and darkcytoplasm, typical to mature RPE cells. The cells in the “SSC high”sorted population were similar to the “Autofluorescent” cells,suggesting that the RPE cells of took over the culture, because of theirhigh proliferation rate. The cells in the “SSC low” were large, hadlight cytoplasm and almost did not proliferate. Some of these cellsgained neuronal-like shapes.

FIGS. 8A-B: Fluorescent microscopy of post-sorting cells of the“Autofluorescent” and the “SSC low” populations, two weeks after thesorting and plating on gelatine. Immunofluorescent staining was carriedout as described in Materials and Methods, using antibodies specific tobestrophin (red) and CRALBP (green). The cells' nuclei werecounterstained with DAPI (blue). The cells in the

“Autofluorescent” sorted population were positively stained with thesemarkers, in contrast with the negative cells in the “SSC low”population. Bestrophin was expressed by 88% of the cells (221 of 251cells) and CRALBP was expressed by 75% of the cells (189 of 251 cells).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof selecting retinal pigmented epithelial cells (RPE cells).

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Current protocols for the derivation of retinal pigmented epithelium(RPE) cells from pluripotent stem cells yields mixed populations ofpigmented and non-pigmented cells. However, pure populations ofpigmented cells are desired for the usage of RPE cells in basicresearch, drug discovery and cell therapy. So far clusters of RPE cellshave been identified as areas of dark pigmentation within cultures ofmultiple subtypes of differentiated cells and were mechanicallydissected and isolated on the basis of their pigmentation. This approachis operator dependent, work intensive, depends on subjective judgment,inconsistent, inaccurate and can not provide pure populations ofpigmented cells.

To overcome these hurdles, the present inventors developed a method forthe purification of RPE cells by fluorescence activated cell sorting(FACS). The present inventors employed FACS sorting to purify RPE cellsfrom mixed-populations of differentiated cells, using novel criteria.The RPE cells were sorted on the basis of their physical properties ofautofluorescence (FIGS. 1A-B) and/or granularity (FIGS. 2A-B), and/or onthe basis of specific membrane markers, such as CD81 (FIG. 3). The cellsafter sorting had a mature RPE polygonal and pigmented phenotype (FIGS.5-7) and expressed RPE specific markers (FIG. 8) as measured byimmunohistochemistry.

Thus, according to one aspect of the present invention there is provideda method of selecting retinal pigmented epithelial (RPE) cells from amixed population of cells, comprising:

(a) analyzing the cells of the mixed population of cells for at leastone of the following parameters:

(i) cells which autofluorescence above a predetermined threshold;

(ii) cells which express CD81 above a predetermined threshold; and

(iii) cells which scatter light perpendicular to a laser beam above apredetermined threshold; and

(b) selecting cells which are positive for at least one of theparameters, thereby sorting RPE cells from a mixed population of cells.

As used herein, the phrase “retinal pigmented epithelial cells (RPE)”refers to cells of a cell type functionally similar to that of nativeRPE cells which form the pigmented cell layer of the retina (e.g. upontransplantation within an eye, they exhibit functional activitiessimilar to those of native RPE cells).

RPE cells may be identified by expression of particular RPE markerproteins—e.g. bestrophin, cellular retinaldehyde-binding protein(CRALBP) retinal pigment epithelium specific protein (RPE65) 65 kDa, RPEtranscription factors microphthalmia-associated transcription factor(MITF) and orthodenticle 2 isoform b homeobox protein (OTX2);tyrosinase, which functions in pigment synthesis; factors that aresecreted by the RPE, such as pigment epithelium-derived factor (PEDF);membrane associated proteins bestrophin, extracellular matrixmetalloproteinase inducer (EMMPRIN), and zona occludens 1 (ZO-1);proteins involved in phagocytosis, including the integrin aV subunit andMer Tyrosine Kinase (MERTK).

According to a particular embodiment, the RPE cells that are selectedexpress at least bestrophin and CRALBP.

According to one embodiment, the RPE cells are native RPE cells of thepigmented layer of the retina and have been removed from donors (e.g.cadavers, see for example Cruz et al., Progress in Retinal and EyeResearch 26 (2007) 598-635; neonates or aborted fetuses, see for exampleAlgvere et al., Graefes Arch Clin Exp Ophthalmol. 1994 December;232(12):707-16). Alternatively, the RPE cells may be somatic cells froma different source (e.g. neuronal cells) which have undergonetrans-differentiation, see for example Opas et al., Int. J. Dev. Biol.45: 633-642 (2001)].

According to another embodiment, the RPE cells are differentiated exvivo from immortalized cell lines.

According to still another embodiment, the RPE cells are differentiatedex vivo from stem cells (SC).

As used herein, the phrase “stem cells” refers to cells which arecapable of remaining in an undifferentiated state (e.g., pluripotent ormultipotent stem cells) for extended periods of time in culture untilinduced to differentiate into other cell types having a particular,specialized function (e.g., fully differentiated cells). Preferably, thephrase “stem cells” encompasses embryonic stem cells (ESCs), inducedpluripotent stem cells (iPS), adult stem cells and hematopoietic stemcells. The stem cells are typically mammalian cells, such as for examplehuman stem cells, rodent stem cells (e.g. mouse or rat) or primate stemcells (e.g. monkey).

The phrase “adult stem cells” (also called “tissue stem cells” or a stemcell from a somatic tissue) refers to any stem cell derived from asomatic tissue [of either a postnatal or prenatal animal (especially thehuman)]. The adult stem cell is generally thought to be a multipotentstem cell, capable of differentiation into multiple cell types. Adultstem cells can be derived from any adult, neonatal or fetal tissue suchas adipose tissue, skin, kidney, liver, prostate, pancreas, intestine,and placenta.

The stem cells utilized by the present invention may also be bone marrow(BM)-derived stem cells including hematopoietic, stromal or mesenchymalstem cells (Dominici, M et al., 2001. Bone marrow mesenchymal cells:biological properties and clinical applications. J. Biol. Regul.Homeost. Agents. 15: 28-37). BM-derived stem cells may be obtained fromiliac crest, femora, tibiae, spine, rib or other medullar spaces.

Mesenchymal stem cells give rise to one or more mesenchymal tissues(e.g., pancreatic, adipose, osseous, cartilaginous, elastic and fibrousconnective tissues, myoblasts) as well as to tissues other than thoseoriginating in the embryonic mesoderm (e.g., neural cells) dependingupon various influences from bioactive factors such as cytokines.Although such cells can be isolated from embryonic yolk sac, placenta,umbilical cord, fetal and adolescent skin, blood and other tissues,their abundance in the BM far exceeds their abundance in other tissuesand as such isolation from BM is presently preferred. Methods ofisolating, purifying and expanding mesenchymal stem cells (MSCs) areknown in the arts and include, for example, those disclosed by Caplanand Haynesworth in U.S. Pat. No. 5,486,359 and Jones E. A. et al., 2002,Isolation and characterization of bone marrow multipotential mesenchymalprogenitor cells, Arthritis Rheum. 46(12): 3349-60.

Preferably, mesenchymal stem cell cultures are generated by diluting BMaspirates (usually 20 ml) with equal volumes of Hank's balanced saltsolution (HBSS; GIBCO Laboratories, Grand Island, N.Y., USA) andlayering the diluted cells over about 10 ml of a Ficoll column(Ficoll-Paque; Pharmacia, Piscataway, N.J., USA). Following 30 minutesof centrifugation at 2,500×g, the mononuclear cell layer is removed fromthe interface and suspended in HBSS. Cells are then centrifuged at1,500×g for 15 minutes and resuspended in a complete medium (MEM, amedium without deoxyribonucleotides or ribonucleotides; GIBCO); 20%fetal calf serum (FCS) derived from a lot selected for rapid growth ofMSCs (Atlanta Biologicals, Norcross, GA); 100 units/ml penicillin(GIBCO), 100 μg/ml streptomycin (GIBCO); and 2 mM L-glutamine (GIBCO).Resuspended cells are plated in about 25 ml of medium in a 10 cm culturedish (Corning Glass Works, Corning, N.Y.) and incubated at 37° C. with5% humidified CO₂. Following 24 hours in culture, nonadherent cells arediscarded, and the adherent cells are thoroughly washed twice withphosphate buffered saline (PBS). The medium is replaced with a freshcomplete medium every 3 or 4 days for about 14 days. Adherent cells arethen harvested with 0.25% trypsin and 1 mM EDTA (Trypsin/EDTA, GIBCO)for 5 min at 37° C., replated in a 6-cm plate and cultured for another14 days. Cells are then trypsinized and counted using a cell countingdevice such as for example, a hemocytometer (Hausser Scientific,Horsham, Pa.). Cultured cells are recovered by centrifugation andresuspended with 5% DMSO and 30% FCS at a concentration of 1 to 2×10⁶cells per ml. Aliquots of about 1 ml each are slowly frozen and storedin liquid nitrogen.

To expand the mesenchymal stem cell fraction, frozen cells are thawed at37° C., diluted with a complete medium and recovered by centrifugationto remove the DMSO. Cells are resuspended in a complete medium andplated at a concentration of about 5,000 cells/cm². Following 24 hoursin culture, nonadherent cells are removed and the adherent cells areharvested using Trypsin/EDTA, dissociated by passage through a narrowedPasteur pipette, and preferably replated at a density of about 1.5 toabout 3.0 cells/cm². Under these conditions, MSC cultures can grow forabout 50 population doublings and be expanded for about 2000 fold[Colter D C., et al. Rapid expansion of recycling stem cells in culturesof plastic-adherent cells from human bone marrow. Proc Natl Acad SciUSA. 97: 3213-3218, 2000].

According to one embodiment, the RPE cells are differentiated frompluripotent stem cells.

The phrase “pluripotent stem cells” as used herein, refers to cellswhich are capable of differentiating into the three embryonic germ celllayers, i.e., endoderm, ectoderm and mesoderm.

According to one embodiment, the pluripotent stem cells compriseembryonic stem cells and/or induced pluripotent stem cells.

The phrase “embryonic stem cells” refers to embryonic cells which arecapable of differentiating into cells of all three embryonic germ layers(i.e., endoderm, ectoderm and mesoderm), or remaining in anundifferentiated state. The phrase “embryonic stem cells” may comprisecells which are obtained from the embryonic tissue formed aftergestation (e.g., blastocyst) before implantation of the embryo (i.e., apre-implantation blastocyst), extended blastocyst cells (EBCs) which areobtained from a post-implantation/pre-gastrulation stage blastocyst (seeWO2006/040763) and embryonic germ (EG) cells which are obtained from thegenital tissue of a fetus any time during gestation, preferably before10 weeks of gestation.

Induced pluripotent stem cells (iPS; embryonic-like stem cells), arecells obtained by de-differentiation of adult somatic cells which areendowed with pluripotency (i.e., being capable of differentiating intothe three embryonic germ cell layers, i.e., endoderm, ectoderm andmesoderm). According to some embodiments of the invention, such cellsare obtained from a differentiated tissue (e.g., a somatic tissue suchas skin) and undergo de-differentiation by genetic manipulation whichre-programs the cell to acquire embryonic stem cells characteristics.According to some embodiments of the invention, the induced pluripotentstem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4and c-Myc in a somatic stem cell.

The embryonic stem cells of the present invention can be obtained usingwell-known cell-culture methods. For example, human embryonic stem cellscan be isolated from human blastocysts. Human blastocysts are typicallyobtained from human in vivo preimplantation embryos or from in vitrofertilized (IVF) embryos. Alternatively, a single cell human embryo canbe expanded to the blastocyst stage. For the isolation of human ES cellsthe zona pellucida is removed from the blastocyst and the inner cellmass (ICM) is isolated by immunosurgery, in which the trophectodermcells are lysed and removed from the intact ICM by gentle pipetting. TheICM is then plated in a tissue culture flask containing the appropriatemedium which enables its outgrowth. Following 9 to 15 days, the ICMderived outgrowth is dissociated into clumps either by a mechanicaldissociation or by an enzymatic degradation and the cells are thenre-plated on a fresh tissue culture medium. Colonies demonstratingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and re-plated. Resulting ES cellsare then routinely split every 4-7 days. For further details on methodsof preparation human ES cells see Thomson et al., [U.S. Pat. No.5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998;Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

According to another embodiment, the ES cells are generated without thedestruction of human embryos—see for example Chung et al Cell Stem cell,vol. 2, no. 2 7 Feb. 2008, pages 113-117.

It will be appreciated that commercially available stem cells can alsobe used with this aspect of the present invention. Human ES cells can bepurchased from the NIH human embryonic stem cells registry(www.escr.nih.gov). Non-limiting examples of commercially availableembryonic stem cell lines are BG01, BG02, BG03, BG04, CY12, CY30, CY92,CY10, TE03 and TE32.

In addition, ES cells can be obtained from other species as well,including mouse (Mills and Bradley, 2001), golden hamster [Doetschman etal., 1988, Dev Biol. 127: 224-7], rat [Iannaccone et al., 1994, DevBiol. 163: 288-92] rabbit [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33], severaldomestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl.43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova etal., 2001, Cloning. 3: 59-67] and non-human primate species (Rhesusmonkey and marmoset) [Thomson et al., 1995, Proc Natl Acad Sci USA. 92:7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

Extended blastocyst cells (EBCs) can be obtained from a blastocyst of atleast nine days post fertilization at a stage prior to gastrulation.Prior to culturing the blastocyst, the zona pellucida is digested [forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA)]so as to expose the inner cell mass. The blastocysts are then culturedas whole embryos for at least nine and no more than fourteen days postfertilization (i.e., prior to the gastrulation event) in vitro usingstandard embryonic stem cell culturing methods.

EG cells are prepared from the primordial germ cells obtained fromfetuses of about 8-11 weeks of gestation (in the case of a human fetus)using laboratory techniques known to anyone skilled in the arts. Thegenital ridges are dissociated and cut into small chunks which arethereafter disaggregated into cells by mechanical dissociation. The EGcells are then grown in tissue culture flasks with the appropriatemedium. The cells are cultured with daily replacement of medium until acell morphology consistent with EG cells is observed, typically after7-30 days or 1-4 passages. For additional details on methods ofpreparation human EG cells see Shamblott et al., [Proc. Natl. Acad. Sci.USA 95: 13726, 1998] and U.S. Pat. No. 6,090,622.

Induced pluripotent stem cells (iPS) (embryonic-like stem cells) can begenerated from somatic cells by genetic manipulation of somatic cells,e.g., by retroviral transduction of somatic cells such as fibroblasts,hepatocytes, gastric epithelial cells with transcription factors such asOct-3/4, Sox2, c-Myc, and KLF4 [Yamanaka S, Cell Stem Cell. 2007,1(1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells fromAdult Mouse Liver and Stomach Cells. Science. 2008 Feb. 14. (Epub aheadof print); I H Park, Zhao R, West J A, et al. Reprogramming of humansomatic cells to pluripotency with defined factors. Nature 2008;451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction ofpluripotent stem cells from adult human fibroblasts by defined factors.Cell 2007; 131:861-872]. Other embryonic-like stem cells can begenerated by nuclear transfer to oocytes, fusion with embryonic stemcells or nuclear transfer into zygotes if the recipient cells arearrested in mitosis.

It will be appreciated that undifferentiated stem cells are of adistinct morphology, which is clearly distinguishable fromdifferentiated cells of embryo or adult origin by the skilled in theart. Typically, undifferentiated stem cells have highnuclear/cytoplasmic ratios, prominent nucleoli and compact colonyformation with poorly discernable cell junctions. Additional features ofundifferentiated stem cells are further described herein under.

Currently practiced ES culturing methods are mainly based on the use offeeder cell layers which secrete factors needed for stem cellproliferation, while at the same time, inhibit their differentiation.Feeder cell free systems have also been used in ES cell culturing, suchsystems utilize matrices supplemented with serum, cytokines and growthfactors as a replacement for the feeder cell layer.

Feeder-Layer Based Cultures

Mouse feeder layers—The most common method for culturing ES cells isbased on mouse embryonic fibroblasts (MEF) as a feeder cell layersupplemented with tissue culture medium containing serum or leukemiainhibitor factor (LIF) which supports the proliferation and thepluripotency of the E S cells [Thomson J A, Itskovitz-Eldor J, Shapiro SS, Waknitz M A, Swiergiel J J, Marshall V S, Jones J M. (1998).Embryonic stem cell lines derived from human blastocysts. Science 282:1145-7; Reubinoff B E, Pera M F, Fong C, Trounson A, Bongso A. (2000).Embryonic stem cell lines from human blastocysts: somaticdifferentiation in vitro. Nat. Biotechnol. 18: 399-404]. MEF cells arederived from day 12-13 mouse embryos in medium supplemented with fetalbovine serum. Under these conditions mouse ES cells can be maintained inculture as pluripotent stem cells, preserving their phenotypical andfunctional characteristics. However, unlike mouse ES cells, the presenceof exogenously added LIF does not prevent differentiation of human EScells. Furthermore, the use of feeder cells substantially increases thecost of production, and makes scale-up of human ES cell cultureimpractical. Additionally, the feeder cells are metabolicallyinactivated to keep them from outgrowing the stem cells, hence it isnecessary to have fresh feeder cells for each splitting of human ESculture. Since at present, the separation of feeder cell components fromembryonic cells prepared in bulk culture cannot be efficiently achieved,feeder cell layer-prepared ES cultures are not suitable for humantherapy.

ES cells can also be cultured on MEF under serum-free conditions usingserum replacement supplemented with basic fibroblast growth factor(bFGF) [Amit M, Carpenter M K, Inokuma M S, Chiu C P, Harris C P,Waknitz M A, Itskovitz-Eldor J, Thomson J A. (2000). Clonally derivedhuman embryonic stem cell lines maintain pluripotency and proliferativepotential for prolonged periods of culture. Dev. Biol. 227: 271-8].Under these conditions the cloning efficiency of ES cells is 4 timeshigher than under fetal bovine serum. In addition, following 6 months ofculturing under serum replacement the ES cells still maintain theirpluripotency as indicated by their ability to form teratomas whichcontain all three embryonic germ layers. Although this system uses abetter-defined culture conditions, the presence of mouse cells in theculture exposes the human culture to pathogens which restricts their usein cell-based therapy.

Human embryonic fibroblasts or adult fallopian epithelial cells asfeeder cell layers—Human ES cells can be grown and maintained usinghuman embryonic fibroblasts, cord blood fibroblasts or adult fallopianepithelial cells. When grown on these human feeder cells the human EScells exhibit normal karyotypes, present alkaline phosphatase activity,express Oct-4 and other embryonic cell surface markers including SSEA-3,SSEA-4, TRA-1-60, and GCTM-2, form teratomas in vivo, and retain all keymorphological characteristics [Richards M, Fong C Y, Chan W K, Wong P C,Bongso A. (2002). Human feeders support prolonged undifferentiatedgrowth of human inner cell masses and embryonic stem cells. Nat.Biotechnol. 20: 933-6].

Foreskin feeder layers—Human ES cells can be cultured on human foreskinfeeder layer as disclosed in U.S. patent appication Ser. No. 10/368,045.Foreskin derived feeder cell layers consist of a complete animal-freeenvironment suitable for culturing human ES cells. In addition, foreskincells can be maintained in culture for as long as 42 passages sincetheir derivation, providing the ES cells with a relatively constantenvironment. Under these conditions the human ES cells were found to befunctionally indistinct from cells grown with alternate protocols (e.g.,MEF). Following differentiation, ES cells expressed genes associatedwith all three embryonal germ layers, in vitro, and formed teratomas invivo, consisting of tissue arising from all three germ layers. Inaddition, unlike human fallopian epithelial cells or human embryonicfibroblasts, human ES cells cultured on foreskin feeder layers weremaintained in culture in a pluripotent and undifferentiated state for atleast 87 passages.

Feeder-Free Cultures

Stem cells can be grown on a solid surface such as an extracellularmatrix (e.g., Matrigel® or laminin) in the presence of a culture medium.

Various methods are known in the art to differentiate embryonic stemcells ex vivo into RPE cells, as summarized in Rowland et al., [JournalCell Physiology, 227:457-466, 2012], incorporated herein by reference.

Two main methods for differentiating hESCs to RPE cells have beendeveloped. One approach is to allow hESCs to overgrow (normally culturedon feeder fibroblasts) and induce spontaneous differentiation throughgrowth factor (i.e. basic fibroblast growth factor—bFGF) removal fromthe maintenance medium.

The hESCs differentiate, overgrow their clean colony borders, and becomemultilayered in this continuous adherent culture method. Timescales varybetween studies and specific cell lines used, but approximately 1-8weeks after growth factor removal, brown-pigmented spots appear. Thesespots develop and expand over time. Approximately 6-14 weeks after thecells are seeded, the cells may be sorted as further described hereinbelow.

The second primary method for differentiating hESC to RPE is throughembryoid body (EB), or neurosphere, formation. According to this method,hESC EBs or neurospheres are generated by passaging colonies to a lowattachment surface (e.g., gelatin or agarose) and culturing theresultant EBs in suspension for a varying amount of time, fromapproximately 1 to 3 weeks or longer, e.g. up to 9 months. After culturein suspension, the aggregates may be seeded on an adherent substrate(e.g., laminin- or poly-D-lysine/fibronectin/laminin-coated plates) andfurther differentiated.

Examples of factors that may be used during the directed differentiationprocess include, but are not limited to WNT antagonists (e.g.,Dickkopf-1 [Dkk-1]) in combination with NODAL antagonists (e.g.,Lefty-A, also a TGF-β ligand). Alternatively, the floating culturedifferentiation media may be supplemented with nicotinamide (vitaminB3), which is thought to stimulate factors in the TGF-b superfamily,which may help pattern RPE during development (Idelson et al., 2010,incorporated herein by reference). Alternatively, the neural inductionmedium may be supplemented with N2 supplement and heparin, or B27, whilethe embryoid bodies are in suspension. Additional factors that may beused include activin A, TGF-β1, and SU5402.

According to a preferred embodiment, the differentiating is effected ina medium comprising at least one member of the transforming growthfactor β (TGFβ) superfamily.

The phrase “transforming growth factor-β (TGFβ) superfamily growthfactor”, as used herein, denotes any member of the TGFβ superfamily ofgrowth factors, such as transforming growth factor-β proteins, includingthe TGFβ1, TGFβ2, and TGFβ3 subtypes, as well as homologous ligandsincluding activin (e.g., activin A, activin B, and activin AB), nodal,anti-mullerian hormone (AMH), some bone morphogenetic proteins (BMP),e.g. BMP2, BMP3, BMP4, BMP5, BMP6, and BMP7, and growth anddifferentiation factors (GDF).

According to a preferred embodiment, the member of the TGFβ superfamilyis preferably the TGFβ1, TGFβ3 growth factors or activin A or acombination of same.

The medium may comprise other components which promote RPE celldifferentiation, such as for example nicotinamide.

Nicotinamide, NA, also known as “niacinamide”, is the amide derivativeform of Vitamin B3 (niacin) which is thought to preserve and improvebeta cell function. NA has the chemical formula C₆H₆N₂O. NA is essentialfor growth and the conversion of foods to energy, and it has been usedin arthritis treatment and diabetes treatment and prevention.

In the context of the present disclosure, the term NA also denotesderivatives of NA.

The term “derivative of nicotinamide (NA)” as used herein denotes acompound which is a chemically modified derivative of the natural NA.The chemical modification may include, for example, a substitution onthe pyridine ring of the basic NA structure (via the carbon or nitrogenmember of the ring), via the nitrogen or the oxygen atoms of the amidemoiety, as well as deletion or replacement of a group, e.g. to form athiobenzamide analog of NA, all of which being as appreciated by thoseversed in organic chemistry. The derivative in the context of theinvention also includes the nucleoside derivative of NA (e.g.nicotinamide adenine). A variety of derivatives of NA are described,some also in connection with an inhibitory activity of the PDE4 enzyme(WO03/068233; WO02/060875; GB2327675A), or as VEGF-receptor tyrosinekinase inhibitors (WO01/55114). For example, the process of preparing4-aryl-nicotinamide derivatives (WO05/014549).

According to an exemplary embodiment the culturing is effected in twostages—the first stage comprises culturing in a medium comprising NA(the medium being devoid of the TGFβ superfamily member) and the secondstage comprises culturing in a medium comprising both the NA and theTGFβ superfamily member (e.g. activin).

The first and the second stage are typically effected for at least twodays, more preferably for about two weeks.

Optionally, the first and second stage are effected in suspension i.e.as freely floating cells, wherein the majority of the cells freely floatin the medium as single cells, as cell clusters and/or as cellaggregates. In other words, the cells survive and propagate in themedium without being attached to a substrate.

According to another embodiment at least one of the stages is performedas an adherent culture (i.e. flat culture) as taught by Vugler et al.,[Exp Neurol. 2008 December; 214(2):347-61. Epub 2008 Sep. 27,incorporated herein by reference].

Following the second stage, the cells are typically cultured for atleast 4 weeks, more preferably, at least 5 weeks, at least 6 weeks, atleast 7 weeks in the same medium but devoid of the TGFβ superfamilymember.

A more detailed method for generating RPE cells from pluripotent stemcells is described in the Examples section below.

Sorting is typically effected no earlier than 4 weeks from the start ofthe directed differentiation protocol, although typically sorting iseffected later i.e. at least one month, at least 6 weeks, at least sevenweeks, at least two months, at least three months, at least four months,at least five months from the start of the differentiation protocol.

Prior to sorting, the generated or isolated cell populations aretypically dispersed using cell dispersing agents. Preferably single cellpopulations are obtained. Examples of agents that may be used todisperse the cells include, but are not limited to collagenase, dispase,accutase, trypsin (e.g. trypsin-EDTA), papain. Alternatively, oradditionally trituration may also be performed to increase the dispersalof the cells.

An exemplary concentration of trypsin that may be used is 0.005-0.1%(e.g. 0.01%) trypsin-EDTA the cells may be incubated with the dispersingagent for about 10-20 minutes, e.g. 15 minutes, at a temperature ofabout 37° C.

It will be appreciated that the mixed cell population from which the RPEcells are selected will comprise different cell types, depending on thesource of the RPE cells. Thus, for example, if the RPE cells areobtained from ex vivo differentiated pluripotent stem cells, the mixedcell population may comprise less differentiated cell types or evennon-differentiated cell types. If the RPE cells are obtained from adonor, the mixed cell population may comprise other retinal cell types.

As mentioned, the RPE cells are selected according to one of thefollowing criteria:

(i) cells which autofluorescence above a predetermined threshold;

(ii) cells which express CD81; and

(iii) cells which scatter light perpendicular to a laser beam above apredetermined threshold.

Selecting cells which express CD81 is typically effected using an agentwhich binds specifically to CD81. Typically, the cells expresssufficient CD81 on their membrane such that they are capable of beingdetected using methods such as FACS, MACS and immunopanning as furtherdescribed herein below.

Typically, the selecting is effected using antibodies that are capableof specifically recognizing this cell-surface protein, although thepresent invention contemplates additional agents such as polynucleotidesor small molecules.

Antibodies which recognize CD81 may be obtained according to methodsknown in the art or may be obtained from commercial sources.

If the CD81 antibody is attached to a magnetic moiety (either directly,or indirectly through a cognate binding molecule), the heterogeneouscell population may be enriched for EpCAM⁺ cells by magnetic activatedcell separation.

If the CD81 antibody is attached is attached to an affinity moiety, theheterogeneous cell population may be enriched for CD81⁺ cells byaffinity purification with the cognate binding molecule. Thus, forexample, if the CD81 antibody is attached to biotin, the heterogenouscell population may be depleted of CD81⁺ cells by purification withstrepavidin beads or column. The CD81⁺ cells can subsequently beretrieved. If, for example the CD81 antibody is attached to an antibodyor an Fc of an antibody, the heterogenous cell population may bedepleted of CD81⁺ cells by purification with protein A beads or column.The CD81⁺ cells can subsequently be retrieved. If the CD81 antibody isattached is attached to a fluorescent moiety, the heterogeneous cellpopulation may be enriched for CD81⁺ cells by using afluorescence-activated cell sorter (FACS).

A Flow Cytometer typically consists of a laser light source, flowmeasurement chamber, and an optical system consisting of lenses,filters, and light detectors. Two photo-multiplier tubes (lightdetectors), one at 180 degrees and one at 90 degrees to the laser, areused to measure forward (FSC) and right-angle scatter (SSC),respectively. Three fluorescence detectors, each consisting of a filterand photomultiplier tube, are used to detect fluorescence. The threedetectors sense green (FL1—530 nm), orange (FL2—585 nm), and redfluorescence (FL3—650 nm). Cells are identified by sort logic applied toall five of the detector signals (FSC, SSC, FL1, FL2, FL3) using acomputer.

Exemplary Flow Cytometers that may be used in this aspect of the presentinvention are manufactured by companies such as Becton Dickinson (USA),Backman Coulter (USA), Partec (Germany).

The FACS machine may be set such that cells of a particular forwardscatter and/or side scatter are selected. Forward-scattered light (FSC)is proportional to cell-surface area or size. FSC is a measurement ofmostly diffracted light and is detected just off the axis of theincident laser beam in the forward direction by a photodiode. FSCprovides a suitable method of detecting particles greater than a givensize independent of their fluorescence.

Side-scattered light (SSC) is proportional to cell granularity orinternal complexity. SSC is a measurement of mostly refracted andreflected light that occurs at any interface within the cell where thereis a change in refractive index. SSC is collected at approximately 90degrees to the laser beam by a collection lens and then redirected by abeam splitter to the appropriate detector.

The present invention contemplates selecting RPE cells using a FACSbased on additional parameters.

For example, the present inventors have shown that by selecting cellsfrom a mixed cell population which autofluoresce using FACS it ispossible to select for RPE cells. According to this embodiment,autofluorescent cells may be sorted using a 780/60 nm filter (FL3-H).The FACS may be set at a voltage of about 429 volts when using thisfilter.

The present inventors have further shown that by selecting cells from amixed cell population which have a high side scatter value using FACS itis possible to select for RPE cells. The FACS may be set at a voltage ofabout 305 volts when testing for this parameter.

It will be appreciated that the present invention contemplates selectingcells based on more than one of the above identified parameters—e.g. twoof the above identified parameters or even all three of the aboveidentified parameters.

Following sorting, the percent of RPE cells in the culture may beexamined—for example by using immunohistochemistry techniques, asdescribed in the Examples section herein below.

RPE cells may be expanded following sorting and optional verification.For expansion, they may be plated at low density on an extra cellularmatrix, preferably poly-D-lysine and laminin, and cultured in serum-freeKOM with NA. Under these culture conditions, the pigmented cells loosepigmentation and acquired a fibroid-like morphology. Following furtherprolonged culture and proliferation into high-density cultures, thecells re-acquired the characteristic polygonal shape morphology andpigmentation of RPE cells. The RPE cells may be expanded in suspensionor in a monolayer. The expansion of the RPE cells in monolayer culturesmay be modified to large scale expansion in bioreactors by methods wellknown to those versed in the art.

Harvesting of the cells may be performed by various methods known in theart. Non-limiting examples include mechanical dissection anddissociation with papain. Other methods known in the art are alsoapplicable.

The sorted RPE cells may be transplanted to various target sites withina subject's eye. In accordance with one embodiment, the transplantationof the RPE cells is to the subretinal space of the eye, which is thenormal anatomical location of the RPE (between the photoreceptor outersegments and the choroids). In addition, dependant upon migratoryability and/or positive paracrine effects of the cells, transplantationinto additional ocular compartments can be considered including thevitreal space, the inner or outer retina, the retinal periphery andwithin the choroids.

Further, transplantation may be performed by various techniques known inthe art. Methods for performing RPE transplants are described in, forexample, U.S. Pat. Nos. 5,962,027, 6,045,791, and 5,941,250 and in EyeGraefes Arch Clin Exp Opthalmol March 1997; 235(3):149-58; BiochemBiophys Res Commun Feb. 24, 2000; 268(3): 842-6; Opthalmic Surg February1991; 22(2): 102-8. Methods for performing corneal transplants aredescribed in, for example, U.S. Pat. No. 5,755,785, and in Eye 1995; 9(Pt 6 Su):6-12; Curr Opin Opthalmol August 1992; 3 (4): 473-81;Ophthalmic Surg Lasers April 1998; 29 (4): 305-8; Ophthalmology April2000; 107 (4): 719-24; and Jpn J Ophthalmol November-December 1999;43(6): 502-8. If mainly paracrine effects are to be utilized, cells mayalso be delivered and maintained in the eye encapsulated within asemi-permeable container, which will also decrease exposure of the cellsto the host immune system (Neurotech USA CNTF delivery system; PNAS Mar.7, 2006 vol. 103(10) 3896-3901).

In accordance with one embodiment, transplantation is performed via parspana vitrectomy surgery followed by delivery of the cells through asmall retinal opening into the sub-retinal space or by direct injection.Alternatively, cells may be delivered into the subretinal space via atrans-scleral, trans-choroidal approach. In addition, directtrans-scleral injection into the vitreal space or delivery to theanterior retinal periphery in proximity to the ciliary body can beperformed.

The sorted RPE cells may be transplanted in various forms. For example,the RPE cells may be introduced into the target site in the form of cellsuspension, or adhered onto a matrix, extracellular matrix or substratesuch as a biodegradable polymer or a combination. The RPE cells may alsobe transplanted together (co-transplantation) with other retinal cells,such as with photoreceptors.

Thus, the invention also pertains to a composition comprising RPE cellsgenerated as described herein and sorted by the method of the invention.The composition is preferably suitable for transplantation into the eye.

The compositions of the present invention may comprise between about20,000-400,000 e.g. 100,000 cells.

Various eye conditions may be treated or prevented by the introductionof the RPE cells obtained by the method of the invention to a subject'seye. The eye conditions may include retinal diseases or disordersgenerally associated with retinal dysfunction, retinal injury, and/orloss of retinal pigment epithelium. A non-limiting list of conditionswhich may be treated in accordance with the invention comprisesretinitis pigmentosa, leber's congenital amaurosis, hereditary oracquired macular degeneration, age related macular degeneration (AMD),Best disease, retinal detachment, gyrate atrophy, choroideremia, patterndystrophy as well as other dystrophies of the RPE, Stargardt disease,RPE and retinal damage due to damage caused by any one of photic, laser,inflammatory, infectious, radiation, neovascular or traumatic injury.

As used herein, the term “treating” or “treatment” refers to thetherapeutic as well as the prophylactic effect of the hSC-derived RPEcells of the invention on a subject's eye condition, the effect maygenerally include, amelioration of symptoms associated with theconditions, lessening of the severity or curing the condition, morespecifically, the effect may include reversal of damage caused to thetreated subject's retina and RPE, improved function of the subject'sretina, rebuilding of the subject's retina and RPE by replacement and/orsupport of failing host retinal and RPE cells, directly or by paracrineeffect as well as to the prophylactic effect which may be exhibited bythe attenuation, inhibition or cessation in damage caused to thesubject's retina as a result of the condition.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 FACS-Sorting Separation of RPE Pigmented Cells from NonPigmented Cells

Materials and Methods

hESC maintenance: hESCs were maintained on human foreskin fibroblaststreated for 2.5 hours with 10 μg/ml mitomycin-C (Sigma, St. Louis, Mo.),and plated in gelatin-coated 9.5 cm² well plates (Nunc, Glostrup,Denmark; 3×10⁵ feeders/well). HES-1 cells were routinely cultured in 85%knockout DMEM medium supplemented with 14% knockout serum replacement,1mM L-glutamine, 1% nonessential amino acids (10 mM of each amino acid),50 U/ml penicillin, 50 μg/ml streptomycin, (all from Gibco, Carlsbad,Calif.) and 4 ng/ml basic fibroblast growth factor (bFGF, Cytolab,Rehovot, Israel). The medium was changed every day. The cells werepassaged weekly as small clusters following digestion with Collagenasetype IV (1 mg/ml, Gibco) for 1 hour.

Differentiation of hESCs to RPE-enriched spheres: HES-1 cells weredifferentiated to spheres, composed of RPE cells and other cell types asdescribed in Idelson et al., Cell Stem Cell, 2009. 5(4): p. 396-408, thecontents of which are incorporated herein by reference. Briefly, hESCcolonies were detached from the tissue culture plates using collagenaseIV (1 mg/ml; Gibco), cultured as floating clusters in knockout medium,comprised of KO-DMEM, 14% KO serum replacement, 1% nonessential aminoacids, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin (allfrom Gibco), and 10 mM nicotinamide (Sigma) in 6-well culture dishes(Costar, Corning Inc., Corning, N.Y.), pretreated with 0.1%low-melting-temperature agarose (FMC BioProducts, Rockland, Me.). Duringthe differentiation procedure, the culture medium was replaced twice aweek. After 2 weeks 140 ng/ml activin A (PeproTech Inc, Rocky Hill,N.J.), was added to the medium for 2 weeks. Then, activin A was omittedfrom the medium and pigmented areas appeared in the floating spheres.The pigmented areas were mature and ready for further analysis orsorting, 7-11 weeks after differentiation was initiated.

Preparation of single cell suspension from mixed spheres: Single cellsuspensions were prepared from 7-11 week spheres using 0.01%trypsin-EDTA (Gibco) treatment for 15 minutes at 37° C. Trypsinizationwas blocked by adding equal volume of DMEM supplemented with 10% fetalcalf serum (Gibco). The cells were then centrifuged at 1200 rpm for 3′,re-suspended in FACS sorting buffer (DMEM without phenol red and withHEPES, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/mlpenicillin, 50 mg/ml streptomycin (all from Gibco), 20 mM glucose and 10mM nicotinamide (both from Sigma). To obtain single-cell suspension, thecells were then filtered through 35 μm filters (Becton-Dickinson,Franklin Lakes, N.J.).

FACS analysis and sorting: FACS analysis was performed using FACSCalibur(Becton-Dickinson) according to standard procedures. FITC-labeledanti-CD81 antibody was purchased from Becton-Dickinson.

Sorting was performed using FACSAria (Becton-Dickinson) through a 100 μmnozzle, at flow rate 1-2, using “purity” mode. Table 1, herein belowsummarizes he voltage values that were used to achieve optimaldefinition of the desired populations:

TABLE 1 Parameter Voltage FSC 3 SSC 305 FL2 (576/26 nm) filter 659 FL3(780/60 nm) filter 429

The mixed population was sorted using the following gates:“autofluorescent” “SSC high” and “SSC low” (shown in FIG. 5).

Analysis of FACS data was carried out using FCS Express (de-novosoftware) and Cellquest (Becton-Dickinson) softwares.

Post sorting RPE cell growth: After the sorting, 1-1.5×10⁵ cells werecentrifuged, re-suspended in DMEM supplemented with 10% fetal calf serum(Gibco) and 10 mM nicotinamide and plated on glass coverslips, that werepre-treated with poly-D-lysine (30-70 KDa, 10 μg/ml; Sigma) and 0.1%porcine-gelatine (Sigma). Cells were grown for 24-48 hours and then themedium was replaced with KO DMEM supplemented with 15% knockout serumreplacement, 1% nonessential amino acids, 2 mM glutamine, 50 U/mlpenicillin, 50 mg/ml streptomycin (all from Gibco), and 10 mMnicotinamide.

Immunofluorescent staining: After 1-2 weeks, the cells were eitherphotographed using standard light microscopy, or fixed with 4%paraformaldehyde, and incubated with primary and secondary antibodiesstained with fluorescent dyes. Nuclei were counterstained with4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame,Calif.). The antibodies used for these stainings were: rabbit antiCRALBP, (Santa Cruz, Calif.), followed by FITC-labeled swine anti-rabbitantibody (DAKO, Glostrup, Denmark) and mouse anti-bestrophin (NovusBiologicals, Littleton, Colo.), followed by Cy3-labelled goat anti mouseantibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.).

Results

To develop protocols for purification of RPE by sorting, the presentinventors initially identified novel markers and properties of RPE cellsthat would distinguish them from other differentiated cells. For thispurpose, differentiation to cultures enriched with pigmented RPE cellswithin a mixture of non pigmented differentiated cells was carried outusing a protocol described in materials and methods. A crude mechanicaldissection was performed that separated the pigmented from thenon-pigmented cell clusters. The two separated cell populations weredisaggregated into single cell suspensions and compared by FACSanalysis. The comparison enabled the identification of the followingmarkers and properties that further allowed sorting of the pigmentedcells:

1. The pigmented cells are auto-fluorescent, relatively to thenon-pigmented cells.

FACS analysis of the pigmented and the non-pigmented clustersdemonstrated auto-fluorescent population in the 780/60 nm filter(FL3-H), which was unique to the pigmented clusters, and did not existwithin the non-pigmented clusters (FIG. 1).

2. The pigmented cells are enriched with a high side scatter population.

FACS analysis of visible light scatter in the direction of the FACSlaser beam (forward scatter, FSC) and perpendicular to the laser beam(side light scatter, SSC), showed that the pigmented clusters areenriched with cells that have high SSC value, in contrast with the cellsfrom the non-pigmented clusters that had relatively low SSC value (FIG.2). These differences in the SSC levels, reflects the disparity in thegranularity of the pigmented and the non-pigmented cells, since RPEcells contain melanin-granules in their cytoplasm. These cytoplasmicgranules turn the laser beam aside, resulting in high SSC values.

3. The pigmented cells express high levels of CD81, relatively to thenon-pigmented cells.

In order to identify extra-cellular markers that may be utilized todistinguish the pigmented from the non-pigmented cells, FACS analysis ofthese cells was performed using antibodies specific to candidateextra-cellular proteins. This analysis showed higher levels of CD81 inthe pigmented population, relatively to the non-pigmented population, asrevealed by their mean fluorescence intensity (FIG. 3).

Single cell suspensions were produced from mixed cell populations andsorted as described in materials and methods. While the pre-sortingpopulation was composed of pigmented and non-pigmented cells, thepost-sorting populations were enriched with pigmented cells. FIG. 4demonstrates the differences between the pre-sort and the post-sortpopulations in one representative experiment. In this experiment, theautofluorescent-gated population was enriched from 8.6% in the pre-sortpopulation to 38.9% at the autofluorescent post-sort population, about a4.5-fold enrichment. Furthermore, the mean fluorescence intensity of theungated cells in the FL3 (780/60 nm) filter was 2-fold higher in theautofluorescent post-sort population (MFI=15) than in the pre-sortpopulation (MFI=7.5), suggesting that the autofluorescent population ishighly enriched following FACS sorting.

After sorting, the cells were plated and grown on 0.1% gelatin-coatedtissue culture plates. After one day, the cells attached to the platesand distinct cell morphologies appeared in the three sorted cell types,illustrated in FIG. 5. In the autofluorescent population, the cells haddark pigmented cytoplasm. In contrast, in the low SSC population, thecells had irregular shapes, a lighter cytoplasm, and no melaningranules. The high SSC population included cells of both types (FIG. 5).

One week later, a typical polygonal RPE morphology was observed in theautofluorescent sorted cells. The cells had dark cytoplasm, resultingfrom pigmented granules (FIG. 6). In contrast, the low SSC cells hadvariable cell morphologies, without cytoplasmic pigmented granules (FIG.6). The progeny of the autofluorescent sorted cells were numerous,relatively to the low SSC sorted cells, indicating a higherproliferation rate of the autofluorescent cells (FIG. 6). The high SSCsorted cells were composed of a mixture of cells, representing both celltypes (FIG. 6).

After 2 weeks in culture, the autofluorescent sorted cells had a matureRPE polygonal and pigmented phenotype, in contrast with the progeny ofthe low SSC sorted cells that almost did not proliferate and had adifferent morphology, of larger and irregular cells with lightcytoplasm. Occasional neurite formation was detected within these cells(FIG. 7). The progeny of the high SSC sorted cells had morphology,similar to the autofluorescent sorted cells, presumably due to rapidproliferation of RPE cells in comparison with the non-RPE cells thatwere in the original high SSC sorted population.

After 2 weeks in culture, the progeny of the sorted populations werefixed and stained with antibodies directed to specific markers of RPEcells, as described in materials and methods. The autofluorescent sortedcells expressed the RPE related markers bestrophin and CRALBP, while thenon-autofluorescent cells did not express these markers (FIG. 8). Thisfinding supports the RPE identity of the sorted autofluorescentpopulation.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A method of selecting retinal pigmentedepithelial (RPE) cells from a mixed population of cells, comprising: (a)analyzing the cells of said mixed population of cells for at least oneof the following parameters: (i) cells which autofluorescence above apredetermined threshold; (ii) cells which express CD81 above apredetermined threshold; and (iii) cells which scatter lightperpendicular to a laser beam above a predetermined threshold; and (b)selecting cells which are positive for at least one of said parameters,thereby sorting RPE cells from a mixed population of cells.
 2. Themethod of claim 1, wherein the RPE cells are characterized by expressionof at least one marker selected from the group consisting of bestrophin,CRALBP, MITF and RPE65.
 3. The method of claim 1, wherein said RPE cellsare generated by ex-vivo differentiating pluripotent stem cells towardsan RPE lineage.
 4. The method of claim 3, wherein said pluripotent stemcells comprise embryonic stem cells (ESCs).
 5. The method of claim 3,wherein said pluripotent stem cells comprise induced pluripotent stem(iPS) cells.
 6. The method of claim 3, wherein said ex vivodifferentiating is effected by culturing said pluripotent stem cells ina medium comprising at least one member of the transforming growthfactor β (TGFβ) superfamily.
 7. The method of claim 6, wherein saidmedium further comprises nicotinamide (NA).
 8. The method of claim 7,wherein said culturing comprises a first culture stage in a first mediumcomprising nicotinamide (NA), said first medium being devoid of said atleast one member of the TGFβ superfamily and a second culture stage in asecond medium comprising NA and said at least one member of the TGFβsuperfamily.
 9. The method of claim 8, wherein said first culture stageis effected for at least two days.
 10. The method of claim 6, whereinsaid at least one member of the TGFβ3 superfamily is selected from thegroup consisting of TGFβ1, TGFβ3 and activin A.
 11. The method of claim1, wherein said selecting cells is performed using a fluorescenceactivated cell sorter (FACS).
 12. The method of claim 11, wherein saidselecting cells which autofluoresce is performed using a 780/60 nmfilter of a FACS.
 13. The method of claim 1, wherein said selectingcells which express CD81 is effected using an antibody which binds toCD81.
 14. The method of claim 13, wherein said antibody comprises adetectable moiety.
 15. The method of claim 1, further comprisingdispersing said mixed population of cells prior to said selecting. 16.The method of claim 15, wherein said dispersing is performed usingtrypsin.
 17. The method of claim 1, further comprising culturing the RPEcells following said selecting in a medium comprising nicotinamide.